Antibiotic contamination of milk following treatment of dairy cows for mastitis or other infections is a major problem in the dairy industry. Beta lactam antibiotics (e.g., penicillins and cephalosporins) are the most common of such contaminants.
Each year, large quantities of milk must be discarded due to antibiotic contamination. Additional economic losses result from the need to recall contaminated milk products and the disruption of milk processing, such as cheese or yoghurt culturing, due to antibiotics in milk.
A substantial portion of the loss due to antibiotic contamination could be avoided if a simple, fast assay for antibiotics in milk could be made at the dairy farm before pickup of the milk by the milk hauler. Detection of contaminated milk at this very early point would prevent contamination of larger quantities of uncontaminated milk in the milk haulers truck and at the milk receiving station.
The current official method for detection of antibiotics in milk relies on the inhibition of bacterial growth by antibiotics. These growth-based assays require a minimum of 21/2 to 3 hours to complete. They are not practical as a means of detecting contaminated milk at the level of the dairy farm or even at the milk receiving station prior to transferring the milk from the hauler's truck to a storage facility.
Several rapid methods have been developed for detecting beta lactam antibiotics in milk. U.S. Pat. Nos. 4,239,745 and 4,239,852 (Charm) describe a rapid method for detecting an antibiotic in a liquid sample based on the competitive binding of the antibiotic contaminant in the sample and a tagged antibiotic to receptor sites on bacterial cells. The commercially-available version of the Charm test requires the use of expensive equipment and a trained operator.
Other proposed rapid assays for antibiotics in milk and other liquids are based on immunochemical reaction and utilize antibodies directed against specific beta-lactam antibiotics. The disadvantages of these assays include (a) the detection of immunochemical reactions requires pretreatment of the sample to remove interfering materials, (b) a mixture of antibodies with specificities for different beta lactam antibiotics is required, (c) rapid detection at 5 to 30 parts per billion requires high affinity, rapidly reacting antibody preparations that are difficult to acquire.
Frere et al ("Enzymatic Method for Rapid and Sensitive Determination of B-Lactam Antibiotics" Antimicrobial Agents and Chemotherapy, October 1980 p. 506-510) describe a rapid enzymatic assay for beta-lactam antibiotics which makes use of the ability of these antibiotics to inactivate a specific D,D-carboxypeptidase produced by the bacterium Actinomadura-R39. Other bacterial D,D-carboxypeptidases are known to be reversibly inhibited by beta-lactam antibiotics, but the R39 enzyme is preferred in that the rate of inactivation is very rapid and the reversal of inhibition is very slow. Thus, over short periods of time, exposure of R39 enzyme to a beta-lactam antibiotic results in a stoichiometric loss of R39 catalytic activity. Measurement of remaining R39 activity after exposure to test samples suspected of containing a beta-lactam antibiotic provides a rapid sensitive assay for the antibiotic.
The assay as described by Frere et al is similar to a commercial test known as Penzym.TM. sold by UCB Bioproducts, Brussels, Belgium. It is rather time-consuming and involves a number of steps and separate reagents. The first step involves an incubation, e.g., five minutes, of the test sample with the carboxypeptidase. If the test sample contains a beta-lactam antibiotic, a certain amount of the enzyme will be inactivated during the incubation depending on the amount of antibiotic present.
The next step involves the addition of a substrate for the carboxypeptidase which is a peptide containing a carboxyterminal D-alanine. This is followed by another incubation (e.g., 15 minutes) during which D-alanine is released from the substrate. Other reagents are added during this incubation period to measure the amount of D-alanine liberated. The liberated D-alanine is oxidized into pyruvic acid by a D-amino acid oxidase enzyme with simultaneous formation of hydrogen peroxide. The hydrogen peroxide oxidizes an organic redox indicator, e.g., o-dianisidine, which provides a colorimetric read-out. Sulfuric acid is added at the end of the incubation period to terminate the reaction and stabilize the color formation. The Penzym.TM. kit is supplied with seven separate reagents including: (1) the D,D-carboxypeptidase; (2) buffer for the D,D-carboxypeptidase; (3) substrate for the D,D-carboxypeptidase ((Acetyl).sub.2 -L-Lys-D-ala-D-ala); (4) flavin adenine dinucleotide, cofactor of the D-amino acid oxidase; (5) peroxidase; (6) o-dianisidine and (7) D-amino acid oxidase.
The Penzym.TM. assay suffers from a number of disadvantages. Firstly, the sequential addition of reagents in several different steps is required. Secondly, the amount of time required to complete the assay, i.e., 20 to 30 minutes, is considered excessive, particularly by milk haulers. Furthermore, an excessive number of separately-packaged reagents must be handled.